We are studying the intracellular metabolism and trafficking of cholesterol in human monocyte-derived macrophages, a major cell type that accumulates cholesterol in atherosclerotic lesions. Human monocyte-macrophages were enriched with cholesterol after incubation with non-lipoprotein cholesterol. The intracellular location of the accumulated cholesterol was studied using electron microscopy and subcellular fractionation of the macrophages. Electron microscopy showed the cholesterol-enriched macrophages contained crystals, (presumably cholesterol crystals), that were surrounded by an electron dense material and bounded by a membrane. Subcellular fractionation studies showed that cholesterol accumulated in at least four distinct cellular compartments (as defined by density). With increasing times of incubation, cholesterol decreased in some compartments and increased in others. This suggests that cholesterol may move between different cellular compartments. Studies using marker enzyme analysis of the gradient fractions has identified one cholesterol-rich compartment as lysosomes. We are continuing work to identify the other cholesterolcontaining cellular compartments and to determine the direction of movement of cholesterol between cellular compartments. Also, experiments will be carried out to determine from what cellular compartment cholesterol leaves monocyte-macrophages during cholesterol efflux.